JP2011256141A - Apoptosis promoter for neutrophil, and anti-inflammatory agent and immunomodulator each promoting apoptosis for neutrophil - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apoptosis promoter for a neutrophil.SOLUTION: The apoptosis promoter for a neutrophil comprises as an effective ingredient a 2-aminophenoxazin-3-one derivative represented by formula I (wherein Rand Rare each independently selected from the group consisting of a hydrogen atom, a 1-4C lower alkyl group, a 1-4C acyl group and a 1-4C acyloxy group) or a pharmacologically acceptable salt or ester thereof.

Description

  The present invention relates to an apoptosis promoter for neutrophils, and an anti-inflammatory agent and an immunomodulator by promoting apoptosis for neutrophils.

Neutrophils are one of the immune cells that generate active oxygen, which is a means for killing bacteria. Active oxygen is, for example, superoxide (O ₂ ⁻ ) and hydrogen peroxide (H ₂ O ₂ ). However, reactive oxygen causes inflammation at the site where neutrophils are activated, and also causes cell damage of cells surrounding the neutrophils as well as cell death of the neutrophils themselves. As a result, the surrounding cells are further destroyed by the release of the cells surrounding the neutrophils as well as the cell contents of the neutrophils themselves, particularly the release of proteolytic enzymes. Accordingly, active oxygen is toxic to the human body (Non-patent Documents 1 and 2 below).

  The neutrophil immune recognition mechanism is based on pattern recognition of non-self substances such as Toll-like receptors. The immune recognition mechanism is inherently retained by human beings. Therefore, immunity by neutrophils is said to be innate immunity. On the other hand, lymphocytes are controlled so that cells that react to specific immunogens (antigens) exposed to the body after birth are selected during the process of development. That is, selectivity for immune recognition of lymphocytes is acquired after birth. Therefore, immunity by lymphocytes is said to be acquired immunity. While neutrophils have a short life span and are consumed (killed) in large quantities, neutrophils are produced in large quantities in the bone marrow and are always supplied in large quantities to the periphery, thus maintaining the number of neutrophil cells in the body. Has been. On the other hand, lymphocytes have a long lifespan, but the number of lymphocytes is controlled by giving growth stimulation only to cells that are selective for stimulation by a specific immunogen (ie, by a growth mechanism according to the environment). Has been. The long-term immune protection against infections once affected is due to the activation of the acquired immune system by long-lived lymphocytes and is also called immune memory. As described above, neutrophils are central immune cells of innate immunity, and lymphocytes are central immune cells of acquired immunity.

Akgut C, Molding DA, Edwards SW.Molecular control of neutrophil apoptosis.FEBS Letters, 487, 318-322 (2001) Yoshida LS, Chiba T, Kakinuma K., Determination of flavin contents in neutrophils by a sensitive chemiluminescence assay: evidence for no translocation of flavoproteins from the cytosol to the membrane upon cell stimulation.Biochim et Biophysica Acta, 1135, 245-252 (1992 )

In order to reduce the amount of active oxygen produced by neutrophils, it is required to promote apoptosis of neutrophils. On the other hand, the influence on other immune cells such as lymphocytes accompanying the promotion of apoptosis must be avoided. Therefore, there is a need for drugs that selectively promote neutrophil apoptosis.
Neutrophils themselves are also involved in pathological conditions related to immune function. Therefore, drugs that promote neutrophil apoptosis are needed for the prevention or treatment of this pathological condition. On the other hand, it is necessary that the drug does not affect other immune cells such as lymphocytes.

  The present invention relates to a pro-apoptotic agent for neutrophils containing a 2-aminophenoxazin-3-one derivative represented by the following formula I or a pharmacologically acceptable salt or ester as an active ingredient, and neutrophil Provided are an anti-inflammatory agent and an immunomodulator by promoting apoptosis on spheres.

Formula I

Here, in Formula I, R ₁ and R ₂ are each independently a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, and an acyloxy group having 1 to 4 carbon atoms. Selected from the group consisting of

  The compound of the above formula I has an antitumor effect (Japanese Patent Laid-Open No. 2-193984 or Japanese Patent Laid-Open No. 2003-2878), has an antiviral effect (Japanese Patent Laid-Open No. 2004-143101), Helicobacter bacteria It has an antibacterial effect against the disease (Japanese Patent Laid-Open No. 2005-060325), has an antibacterial effect against chlamydia (Japanese Patent Laid-Open No. 2005-272334), and is effective against type 2 diabetes (Japanese Patent Laid-Open No. 2006-089399) Publication) is known. However, the present inventors have found for the first time that the compound of the above formula I has an apoptosis promoting effect on neutrophils.

The agent of the present invention promotes apoptosis of neutrophils, and as a result, exhibits an anti-inflammatory effect and an immunomodulatory effect. On the other hand, the agent does not exhibit cytotoxicity against lymphocytes and monocytes. Therefore, the agent has a very high selectivity for neutrophils in promoting apoptosis. Further, as described above, neutrophils are the central immune cells of innate immunity. Therefore, according to the selectivity, diseases caused by innate immunity are effectively treated with the agent of the present invention.
Also, activated neutrophils release inflammatory cytokines such as IL-1, IL-6, TNFα, IL-8, G-CSF, and GM-CSF. These cytokines decrease cytotoxic T cells and helper T cells, and increase suppressor T cells. Therefore, when these cytokine concentrations increase due to abnormal survival of activated neutrophils, cellular immunity is suppressed and various diseases are caused for the living body. Therefore, the function of cytotoxic T cells and helper T cells can be maintained by apoptosis of activated neutrophils with the agent of the present invention, and the cellular immunity is maintained.

It is a photograph of lymphocytes, neutrophils and monocytes treated with a phenoxazine compound. It is a photograph of lymphocytes and neutrophils in an additive-free negative control and a solvent negative control. It is a graph which shows the appearance rate of the dead cell of a neutrophil. It is a graph which shows the appearance rate of the dead cell of a lymphocyte.

The agent of the present invention contains a 2-aminophenoxazin-3-one derivative represented by the following formula I or a pharmacologically acceptable salt or ester as an active ingredient. In formula I, R ₁ and R ₂ are each independently a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms, and an acyloxy group having 1 to 4 carbon atoms. It is arbitrarily selected from the group. Preferably, R ₁ and R ₂ are each independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, hydroxymethyl, hydroxyethyl, methoxy, ethoxy, methoxymethyl and ethoxymethyl groups. Selected. More preferably, R ₁ is a hydrogen atom or a methyl group, R ₂ is a hydrogen atom or a methyl group, and more preferably R ₂ is a hydrogen atom.

Formula I

  In the present invention, “promotion of apoptosis for neutrophils” means promoting apoptosis of neutrophils, inducing apoptosis in neutrophils, and / or making neutrophils reach apoptosis earlier. Say.

  The “apoptosis-promoting agent” of the present invention is used to treat active oxygen produced by neutrophils or proteolytic enzymes released from neutrophils, particularly for diseases that require promotion of neutrophil apoptosis. It can be used for the disease caused. Furthermore, the apoptosis promoting agent of the present invention can be used for diseases caused by inflammatory cytokines released by neutrophils. Examples of such diseases include systemic inflammatory syndrome (SIRS), acute respiratory distress syndrome (ARDS), ischemia reperfusion disorder, rheumatoid arthritis, ulcerative colitis, gangrene Examples include, but are not limited to, pyoderma, Sweet syndrome, and Behcet's disease. The “apoptosis promoter” of the present invention can also be used in various tests or tests that require the promotion of neutrophil apoptosis.

  The “anti-inflammatory agent” of the present invention is an inflammatory disease in which apoptosis of neutrophils is required, in particular, an inflammatory disease caused by innate immunity, more particularly reactive oxygen produced by neutrophils or It can be used against inflammatory diseases caused by proteolytic enzymes released from neutrophils or inflammatory diseases caused by neutrophils themselves. Examples of such diseases include dermatitis associated with increased neutrophils, enteritis associated with increased neutrophils (gastrointestinal inflammatory diseases), oral cavity / gingivitis, pneumonia, pancreatitis, nephritis, hepatitis, encephalomyelitis, Inflammation associated with neoplastic diseases, inflammation resulting from infection or metabolic disorders, inflammation associated with tissue necrosis such as burns or frostbite, other inflammatory diseases associated with increased neutrophils, autoimmune diseases or allergic diseases However, it is not limited to these. That is, the anti-inflammatory agent of the present invention can be used for the prevention or treatment of these diseases.

  The “immunomodulator” of the present invention is an immune-related disease that requires promotion of apoptosis of neutrophils, in particular, a disease caused by innate immunity, and more particularly, regulation of immune function by causing apoptosis in neutrophils Can be used for diseases in need. Examples of such diseases include autoimmune diseases, allergic diseases, transplant rejection after organ transplantation, or non-biological materials (medical devices or medical materials such as vascular stents, heart catheters, bones, teeth, etc.). Although a post-planting reaction can be mentioned, it is not limited to these. That is, the immunomodulator of the present invention can be used for the prevention or treatment of these diseases.

  Furthermore, the agent of the present invention is used as a set with other agents. Anti-inflammatory agents other than the agents of the present invention, immunomodulators, apoptosis promoters, anticancer agents, anti-HIV agents, antibiotics, antifungal agents, enzyme agents, enzyme inhibitors, antioxidants, lipid uptake Inhibitor, hormone agent, immunostimulator, protease inhibitor, reverse transcriptase inhibitor, human growth hormone, steroid agent, vasodilator, angiotensin converting enzyme inhibitor, angiotensin receptor antagonist, smooth muscle cell proliferation / migration Inhibitors, platelet aggregation inhibitors, anticoagulants, chemical mediator release inhibitors, vascular endothelial cell proliferation or inhibitors, aldose reductase inhibitors, mesangial cell proliferation inhibitors, lipoxygenase inhibitors, Maillard reaction inhibitors, Examples include amyloidosis inhibitors, NOS inhibitors, AGEs inhibitors, radical scavengers, and the like. In particular, the agent of the present invention can be used in a set (combination) with an agent used for treatment of a patient having an inflammatory disease or an immune disease.

  The agent of the present invention is used for humans or animals (pets such as dogs and cats; livestock such as cows, horses and pigs; poultry such as chickens; fish such as tuna and sea breams).

  Salts that can be used in the agent of the present invention include, for example, inorganic salts such as alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts; metal salts such as copper salts and zinc salts; , Diethanolamine salts, alkanolamine salts such as 2-amino-2-ethyl-1,3-propanediol salt and triethanolamine salt; heterocyclic amine salts such as morpholine salt, piperazine salt and piperidine salt; ammonium salt and arginine salt , Basic amino acid salts such as lysine salt and histidine salt can be mentioned, but not limited thereto. Here, the basic amino acid may be D-form, L-form, or a mixture thereof.

  Examples of the ester that can be used in the agent of the present invention include, but are not limited to, formic acid ester, acetic acid ester, and propionic acid ester.

  The compound represented by formula I is preferably produced according to the methods described in JP-A Nos. 02-193984 and 2003-2878, but is not limited thereto.

  The agent of the present invention can be used in the form of a general pharmaceutical preparation. In the preparation, diluents such as fillers, fillers, binders, moistening agents, disintegrating agents, surfactants, lubricants and the like that are usually used, or excipients can be blended. Various forms of the agent of the present invention can be selected according to the purpose of treatment, and representative examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, and liquids. Injections such as suspensions, eye drops, or ointments.

  Various carriers that are conventionally used can be used as carriers to be formed into tablets. For example, excipients such as lactose, sucrose, glucose, sodium chloride, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid; water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxy Binders such as methylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone; dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid Disintegrating agents such as monoglyceride, starch and lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter and hydrogenated oil; absorption accelerators such as quaternary ammonium base and sodium lauryl sulfate, glycerin Humectants such as starch, lactose, kaolin, bentonite, colloidal silicic acid and the like; lubricants such as purified talc, stearate, boric acid powder and polyethylene glycol can be used, but are not limited thereto . Further, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

  When forming into the form of a pill, a carrier conventionally known in this field can be widely used. For example, excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin and talc; binders such as gum arabic powder, tragacanth powder, gelatin and ethanol; disintegrants such as laminaran and agar can be used. .

  Capsules are usually prepared by mixing an active ingredient compound with the various carriers exemplified above and filling them into hard gelatin capsules, soft capsules and the like according to a conventional method.

  Conventionally known carriers can be widely used for forming into a suppository form. Examples thereof include polyethylene glycol, cacao butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like.

  When prepared as injections, solutions, emulsions and suspensions are preferably sterilized and isotonic with blood. In molding into these forms, any conventionally used diluent in this field can be used. For example, water, ethanol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used, but are not limited thereto. In this case, a sufficient amount of sodium chloride, glucose or glycerin may be contained in the pharmaceutical preparation to prepare an isotonic solution, and a normal solubilizing agent, buffering agent, soothing agent, etc. may be added. May be.

  When prepared as an ointment, oily bases conventionally known in this field can be widely used. For example, peanut oil, sesame oil, soybean oil, safflower oil, avocado oil, sunflower oil, corn oil, rapeseed oil, menthol oil, sunflower oil, camellia oil, palm oil, olive oil, poppy oil, cacao oil, beef tallow, lard Oils such as wool oil; mineral oils such as petrolatum, paraffin, silicon oil, squalane; isopropyl myristate, n-butyl myristate, isopropyl linoleate, acetyl ricinolate, stearyl ricinolate, diethyl sebacate, diisopropyl adipate, Higher fatty acid esters such as cetyl alcohol, stearyl alcohol, white beeswax, whale wax and tree wax, higher fatty alcohols and waxes, higher fatty acids such as stearic acid, oleic acid and palmitic acid; saturated or having 12 to 18 carbon atoms Monounsaturated fatty acids , Di -, a triglyceride mixture. In the agent of this invention, you may use these bases individually by 1 type or in mixture of 2 or more types.

  In the agent of the present invention, conventional additives such as metal soaps, animal / plant extracts, vitamins, hormones, amino acids and other medicinal agents, surfactants, dyes, dyes, pigments, fragrances, UV absorbers, moisturizing agents An agent, a thickener, an antioxidant, a sequestering agent, and a pH adjuster can be appropriately blended as necessary.

  The blending amount of the agent of the present invention is not particularly limited as long as it has its efficacy effect, but is usually about 0.0001 to 10% by weight, preferably 0.001 to 10% by weight in the composition containing the agent of the present invention. The amount is preferably about 0.01 to 10% by weight, more preferably about 0.1 to 5% by weight.

  The method of using the agent of the present invention is not particularly limited, and it is used in a method according to various preparation forms, age of patients or users, sex and other conditions, degree of disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules can be administered orally. In the case of liquids, sprays, aerosols, emulsions, and ointments, they can be administered nasally or by eye drops. Further, in the case of a spray or aerosol, it can be administered by inhalation. In the case of an injection, it can be administered alone or mixed with a normal replacement fluid such as glucose or amino acid and intravenously administered, and if necessary, it can be administered intramuscularly, intradermally, subcutaneously or intraperitoneally. In the case of a suppository, it can be administered rectally. In the case of an external preparation, it can be applied to the affected area.

  The amount of the agent of the present invention is appropriately selected depending on the usage, the subject patient or animal, the age of the user or animal, gender and other conditions, the degree of disease, etc. The daily weight per kg is preferably about 1 to 20 mg, and is preferably divided into about 1 to 3 times a day.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these Examples.

The phenoxazine compounds used in the examples were 2-amino-4,4α-dihydro-4α-7-dimethyl-3H-phenoxazin-3-one (hereinafter referred to as “Phx-1”) and 2-amino-. Phenoxazin-3-one (hereinafter referred to as “Phx-3”). In Phx-1, in Formula I, R ₁ is a methyl group and R ₂ is a hydrogen atom. In Phx-3, in Formula I, R ₁ and R ₂ are both hydrogen atoms. Phx-1 and Phx-3 are shown below in formulas II and III, respectively.

Formula II

Formula III

  As a phenoxazine compound of Comparative Example, 3-amino-1,4α-dihydro-4α-8-dimethyl-2H-phenoxazin-2-one (hereinafter referred to as “Phx-2”) was used. Phx-2 is shown in Formula IV below.

Formula IV

  Phx-1 and Phx-3, and Phx-2 were produced by the above-described method using bovine hemoglobin (Japanese Patent Laid-Open No. 2003-2878, Shimizu S, Suzuki M, Tomoda A, Arai S, Taguchi H, Hanawa T, Kamiya S. Phenoxazine compounds produced by the reactions with bovine hemoglobin showantimicrobial activity against non-tuberculosis mycobacteria.Tohoku J Exp Med. 2004; Volume 203: 47-52, Tomoda A, Arai S, Ishida R, Shimamoto T, Ohyashiki K. An improved method for the rapid preparation of 2-amino-4,4α-dihydro-4α-7-dimethyl-3H-phenoxazine-3-one, a novel antitumor agent. Biorg Med Chem Lett. 2002 11: 1057-1058).

Preparation of phenoxazine solution The three phenoxazine compounds produced as described above were each dissolved in 3: 1 (volume ratio) dimethyl sulfoxide (DMSO) and ethyl alcohol to prepare a 10 mM solution.

5 ml of blood was collected in a sterile blood collection glass tube containing heparin (Benoject II vacuum blood collection tube, Terumo Corporation) from healthy adults who had obtained consent to collect blood . Half of the heparinized blood was subjected to the following blood culture test within 1 hour after blood collection. Plasma was obtained from the remaining blood by centrifugation (3000 rpm, 10 minutes). The plasma was stored at 4 ° C. and used in the blood reconstitution described below.

Blood culture test
55 ml FBS (deactivated) (Fetal Bovine Serum, JR Scientific, Inc.) was added to 500 ml RPMI-1640 (RPMI-1640 Medium, Sigma). After addition of FBS, 900 μl of the RPMI-1640 was added to a 24-well culture plate (IWAKI Non-treated Microplate, ASAHI TECHNO GLASS). Next, 10, 5 or 2.5 μl of 10 mM Phx-1, Phx-2 or Phx-3 was added. After thoroughly stirring the solution in each well, 100 μl of the above heparinized blood was added to each well and further stirred. The final concentration of each phenoxazine compound was 100 μM, 50 μM, or 25 μM for each of the added amounts of 10, 5 or 2.5 μl.
Next, the culture solution in the well was cultured at 37 ° C. for 18 hours in a humidified atmosphere containing 5% CO ₂ . Immediately after completion of the culture, the culture solution was stirred, 1 ml of the culture solution was transferred to a 1.5 ml Eppendorf tube, and centrifuged to precipitate blood cells (including red blood cells, white blood cells, lymphocytes and platelets). After removing the supernatant plasma, 50 μl of the plasma stored at 4 ° C. was added to the precipitated blood cells to reconstitute the blood. Next, the reconstructed blood was applied to a slide glass to prepare a blood smear specimen. The slide glass was air-dried, fixed with methanol, and Diff-Quick staining (Diffquick staining solution I, Diffquick staining solution II, Sysmex International Reagents Co., Ltd.) was prepared. As a control group, a negative control without addition and a solvent negative control to which DMSO-ethyl alcohol (3: 1 (volume ratio)) solvent was added at 10 μl / ml were prepared, and stained specimens were also prepared. For these stained specimens, morphological changes of neutrophils, monocytes and lymphocytes were observed under a microscope (300 times). When a morphological change was observed, the appearance rate of the change (appearance rate of dead cells) was expressed in percentage.

Results FIG. 1 is a photograph of the results of treatment of lymphocytes, neutrophils and monocytes with 100 μM Phx-1, Phx-2 or Phx-3. FIG. 2 is a photograph showing the results for the non-added negative control and the solvent negative control.
When treated with Phx-1, nuclear condensation, chromatin staining and cytoplasmic vacuole formation were observed in neutrophils (FIG. 1). Even when treated with Phx-3, nuclear condensation, chromatin staining and cytoplasmic vacuole formation were observed in neutrophils (FIG. 1). These phenomena indicate that neutrophil apoptosis was induced by Phx-1 or Phx-3.
When treated with Phx-2, the above phenomenon was not observed (FIG. 1). The above phenomenon was not observed in the negative control without addition and the solvent negative control (FIG. 2).
In addition, when treated with Phx-1 or Phx-3, no morphological changes in monocytes and lymphocytes were observed (FIG. 1). This indicates that Phx-1 and Phx-3 do not cause apoptosis in monocytes and lymphocytes. That is, Phx-1 and Phx-3 are not cytotoxic to these cells.

  Table 1 shows the number of live and dead neutrophil cells and the appearance rate of dead cells in the smear specimen. In counting these cells, one or two smear samples were used so that the total number of cells exceeded 200. In addition, in order to suppress the dispersion | variation by the position on a smear sample, the said count was implemented while moving so that it may not count the same position of a smear sample by making it into 1 unit from the start of a smear pull to the end. FIG. 3 shows a graph of the dead cell appearance rate.

When treated with Phx-1 or Phx-3, respectively, the number of dead cells was higher compared to the no-additive negative control and the solvent negative control. In addition, when treated with Phx-1 or Phx-3, the number of dead cells was greater than when treated with Phx-2. These results indicate that Phx-1 and Phx-3 have a pro-apoptotic effect.
In addition, the number of dead cells when treated with Phx-1 or Phx-3 increased in a concentration-dependent manner. That is, the apoptosis promoting effect by Phx-1 and Phx-3 is concentration-dependent.
Furthermore, when treated with Phx-1, the number of dead cells was greater than when treated with Phx-3. That is, Phx-1 has a stronger apoptosis promoting effect than Phx-3.
Phx-2 showed a slight pro-apoptotic effect at a concentration of 100 μM, but did not show a pro-apoptotic effect at concentrations of 25 μM and 50 μM, as did the negative control and the solvent negative control.

  Table 2 shows the number of living and dead cells of lymphocytes and the appearance rate of dead cells in the smear specimen. These cell counts were performed as described above. FIG. 4 shows a graph of the dead cell appearance rate.

  Phx-2 and Phx-3 did not show cytotoxicity against lymphocytes in human peripheral blood. Phx-1 was slightly cytotoxic to lymphocytes in human peripheral blood only when the concentration of the solution was 100 μM (Table 2).

Claims (9)

Formula I
Wherein R ₁ and R ₂ are each independently a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms. A pro-apoptotic agent for neutrophils, which comprises a 2-aminophenoxazin-3-one derivative or a pharmacologically acceptable salt or ester thereof as an active ingredient. R ₁ and R ₂ are each independently selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a hydroxymethyl group, a hydroxyethyl group, a methoxy group, an ethoxy group, a methoxymethyl group, and an ethoxymethyl group. The apoptosis promoting agent according to claim 1, which is selected. The apoptosis promoting agent according to claim 1 or 2, wherein R ₁ is a hydrogen atom or a methyl group, and R ₂ is a hydrogen atom. Formula I
Wherein R ₁ and R ₂ are each independently a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms. And an anti-inflammatory agent by promoting apoptosis against neutrophils, comprising as an active ingredient a 2-aminophenoxazin-3-one derivative or a pharmacologically acceptable salt or ester selected from the group consisting of . R ₁ and R ₂ are each independently selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a hydroxymethyl group, a hydroxyethyl group, a methoxy group, an ethoxy group, a methoxymethyl group, and an ethoxymethyl group. The anti-inflammatory agent according to claim 4, which is selected. The anti-inflammatory agent according to claim 4 or 5, wherein R ₁ is a hydrogen atom or a methyl group, and R ₂ is a hydrogen atom. Formula I
Wherein R ₁ and R ₂ are each independently a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, an acyl group having 1 to 4 carbon atoms and an acyloxy group having 1 to 4 carbon atoms. And an immunoregulatory agent by promoting apoptosis against neutrophils, comprising as an active ingredient a 2-aminophenoxazin-3-one derivative or a pharmacologically acceptable salt or ester selected from the group consisting of . R ₁ and R ₂ are each independently selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a hydroxymethyl group, a hydroxyethyl group, a methoxy group, an ethoxy group, a methoxymethyl group, and an ethoxymethyl group. The immunomodulator according to claim 7, which is selected. The immunomodulator according to claim 7 or 8, wherein R ₁ is a hydrogen atom or a methyl group, and R ₂ is a hydrogen atom.